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In 1985, I was accepted as postdoc by Professor Forte of UC Berkeley. He discovered H + ,K + -ATPase and established the membrane recycling theory as the activation mechanism for acid secretion using whole animals. H + ,K + -ATPase is an enzyme that exchanges H + with K + . In resting state, it locates on the tubulovesicles and the pump does not work because the membrane lacks K + permeability. Upon stimulation, the tubulovesicles fuse to the apical membrane and acquire K + permeability, turning the pump on. The main route was known to be protein kinase A (PKA), but its specific targets remained unknown. Right after I joined Forte's lab, I was fortunate enough to reproduce the above mechanism in vitro, and I discovered proteins of molecular weight 120 kDa and 80 kDa that were specifically phosphorylated in stimulated parietal cells. After I returned to Japan, the former was cloned and named as parchorin, which is one of the chloride intracellular channels. Although no progress was made on ezrin, I found out the importance of PIP 2 and Arf6 using permeabilized gland models. After I left parietal cell research, the link between ezrin and Arf6 was revealed. PKA phosphorylates S66 of ezrin and also MST4. The former changes the N-terminal structure of ezrin to bind syntaxin3, and the latter phosphorylates ACAP4, an Arf6-GAP, to accelerate binding to ezrin. Subsequently, H + ,K + -ATPase, SNAREs, ezrin, and Arf6-GAP are aligned on the apical membrane. However, there are still many unsolved questions and the intracellular mechanism of parietal cells remains an attractive research area.

It-is well known that dopamine is necessary for motor function, reward-motivated behaviors. The principal target of dopamine is medium spiny neurons (MSNs), which are a special type of GABAergic inhibitory cell that represents,90% of the neurons within the striatum, including the nucleus accumbens (NAc). There is a distinct class of spatially intermixed MSNs that express dopamine type 1 or 2 receptors (DlR-MSNs or D2R-MSNs, respectively). The D1R-MSN and D2R-MSN pathways control the dynamic balance in the basal ganglia circuit. D1R is coupled -to adenylate cyclase through Golf to activate protein kinase A (PKA), whereas D2R inhibits adenylate cyclase through Gi. Although PKA has been implicated in reward signals downstream of D1Rs by pharmacological approaches, there is no direct evidence indicating that PKA in DlR-MSNs regulates neuronal excitability and reward-related behaviors. We have established the system in which enzymatic activity can be manipulated in the NAc under the control of specific promoters for DlR-MSNs using adeno-associated virus (AAV)-mediated conditional'transgenic techniques. We have also found novel PKA substrates in dopamine receptor signaling using phosphoproteomic approach. This review article focuses on the PKA and its substrates to understand dopamine-related signals in the MSNs. We discuss phosphoproteomic approaches for' comprehensively screening of dopamine signaling. Finally, we highlight RapI pathway as a novel reward signal in vivo.

Upon elevation of plasma glucose concentration, pancreatic β-cells generate bursts of action potentials to induce cyclic changes in [Ca(2+)]i regulating insulin release. Glucose-dependent insulin secretion is synergistically enhanced by glucagon-like peptide-1 (GLP-1), which increases [cAMP]i and activates protein kinase A (PKA) and exchange protein activated by cAMP (Epac). The insulinotropic effect of GLP-1 is mediated, at least in part, by modulating multiple ion channels/transporters at the plasma membrane and ER through PKA- and EPAC-dependent mechanisms, which increase membrane excitability and intracellular Ca(2+) release. However, because of complex interactions between multiple cellular factors involved in the GLP-1 effects, quantitative aspects of the molecular/ionic mechanisms have not yet been determined. We thus performed simulation studies and mathematical analysis to investigate how GLP-1 signals control [cAMP]i and subsequently modify the bursting activities and Ca(2+) dynamics. First, a GLP-1 receptor signal transduction model was developed and introduced to our β-cells model. Secondly, modulatory effects of PKA/Epac on ion channels/transporters were incorporated based on experimental studies. Increases in the frequency and duration of the bursting activity observed during GLP-1 stimulation were well reconstructed by our model, and lead potential analysis quantitatively determined the functional role of each ion channel/transporter in modifying the burst pattern. Finally, an IP3R model was developed to reproduce GLP-1-induced Ca(2+) transients/oscillations. Instantaneous equilibrium analysis and bifurcation analysis also elucidated the quantitative mechanisms involved in generating IP3R-mediated Ca(2+) mobilization. The results of this theoretical analysis of the effects of GLP-1 on membrane excitability/Ca(2+) dynamics are discussed in this review.

Parathyroid hormone-related peptide (PTHrP) is known as an important local factor for chondrogenesis, promoting chondrocyte proliferation and inhibiting their differentiation into the hypertrophic phenotype. Signaling transduction through the PTH/PTHrP receptor has two possible pathways: the activation of adenylate cyclase and subsequent protein kinase A (PKA), and the activation of phospholipase C (PLC). Recent studies with mice carrying PTH/PTHrP receptor inactivated for PLC and chondrocyte-specific deletion of the G (s) gene have shown that cAMP/PKA signaling appears to stimulate chondrocyte proliferation and inhibit their differentiation, whereas PLC signaling enhanced chondrocyte differentiation and inhibited their proliferation. In a physiological state, cAMP/PKA signaling may predominate over PLC pathway. Also, Na(+)/H(+)exchanger regulatory factor 2 (NHERF2) has been reported to down-regulate adenylate cyclase activity, in a switch mechanism that results in signal transduction through the PLC pathway.

Both metformin and thiazolidinedione derivatives(TZDs) improve insulin resistance, a major pathogenesis of type 2 diabetes, and decrease blood glucose levels without stimulating insulin secretion. Metformin inhibits glucose output from the liver, while TZDs increase glucose utilization in the peripheral tissues. In addition, there has been indicated that these agents ameliorate metabolic syndrome beyond glucose-level lowering. Molecular targets of these agents have recently been revealed; AMP-activated protein kinase (AMPK) for metformin and adiponectin, while PPAR gamma for TZDs which induce gene expression of adipocyte glycerol kinase and adiponectin. Insulin-sensitizing agents are clinically useful for obese diabetic patients with insulin resistance. However, periodical examinations are necessary to avoid serious adverse effects such as lactic acidosis, although rare, by metformin and liver injury by TZDs.

Nootropics are proposed to serve as cognition enhancers. The underlying mechanism, however, is largely unknown. We have attempted to assess the intracellular signal transduction pathways mediating the action of nefiracetam, a nootropic agent, on neuronal Ca2+ channels and nicotinic ACh receptors. In NG108-15 cells, nefiracetam (1 microM) enhanced the activities of N/L-type Ca2+ channels without affecting T-type The nefiracetam action was mimicked by dibutyryl cAMP (1 mM), or blocked by pertussis toxin (PTX), indicating that PTX-sensitive inhibitory G-proteins and cAMP-dependent pathways mediate the drug action. Nefiracetam also exerted a dose-dependent biphasic effect on Torpedo nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes, in which the drug induced a short-term depression of ACh-evoked currents at submicromolar concentrations (0.01-0.1 microM) and a long-term enhancement of the currents at micromolar concentrations (1-10 microM). The depression was caused by activation of PTX-sensitive G-protein-regulated cAMP-dependent protein kinase (PKA) with subsequent phosphorylation of the ACh receptors; in contrast, the enhancement was caused by activation of Ca(2+)-dependent protein kinase C (PKC) and the ensuing PKC phosphorylation of the receptors. It is concluded that nefiracetam interacts with PKA and PKC pathways, which may explain a cellular mechanism for the action of cognitive enhancers.

The genetic basis of myotonic dystrophy (DM) is an unstable expansion of CTG repeats that encodes a putative protein kinase A (PKA). Expanding CTG repeats length correlates central nervous involvements and cardiac disorders. Moreover, there are studies combining endocrine abnormalities and molecular analysis. In these reports, both the urinary excretion of phosphate in the Ellsworth-Howard test and serum TSH response in the thyrotropin releasing hormone (TRH) tolerance test correlate with size of CTG expansion. The Ellsworth-Howard test and the TRH tolerance test have been postulated to be mediated through the PKA and protein kinase C (PKC), respectively, suggest that the DM gene relates to a PKA or one part of the PKA pathway which influences the PKC pathway.

It has been known that the stimulation of skin cells by physiologically active substances is accompanied by the quantitative and qualitative alterations of the glycosaminoglycans (GAGs) in skin. This phenomenon make it possible to evaluate the activity of test substances for the stimulation of skin cells. The purpose of our study is to elucidate the effectiveness of inorganic compounds for the stimulation of skin cells. (1) The cultured skin cells including keratinocytes and fibroblasts, (2) the cultured model skin constituted of collagen gel, fibroblasts and keratinocytes, and (3) the mouse damaged skin were used in the present study. The results obtained from these studies demonstrate that inorganic substances commonly have a possibility to activate cyclic AMP-dependent protein kinase (A-kinase), and the A-kinase activation results in an increase in GAGs. These findings brought the scientific basis for the effectiveness of the Japanese traditional balneotherapy for damaged skin.

G proteins serve as transducers between cell surface receptors and intracellular effectors. They consist of three subunits, termed alpha, beta, and gamma. Recently, it has been recognized that the beta gamma subunits play an active role such as activation of beta-adrenergic receptor kinase (beta ARK). The desensitization and down-regulation of beta-adrenergic receptors have been observed in the heart failure. beta ARK is one of the components involved in desensitization of beta-adrenergic receptor and it is reported, recently, that G protein beta gamma subunits bind beta ARK through the pleckstrin homology domain. Therefore, we investigated the effects of beta-adrenergic receptor stimulation on steady-state level of G protein beta subunits (G beta) in the rat heart. The whole rat heart was preliminarily perfused for 10 min by Langendorff's technique at 60 mmHg of hydrostatic pressure with Krebs-Henseleit bicarbonate buffer, and then perfused for 30 min in the same buffer with or without 10 microM isoproterenol (ISO), 0.1mM epinephrine (EPI), 10 microM ISO with 0.1mM propranolol (PROP), or 10 microM ISO with 10 microM CGP20712A (CGP). Immunoblotting using isoform-specific antisera against G protein beta subunits revealed that the rat heart contains at least three G protein beta subunits, beta 1, beta 2 and beta 3 at molecular weight of between 35,000 and 37,000. The level of G beta 3 in the cytosol dramatically decreased in the presence of ISO alone or ISO with CGP. G beta 3 decreased in the presence of EPI as well. Propranolol could block ISO-induced decrease of G beta 3 in the cytosol. In contrast, the levels of G beta 1 and G beta 2 didn't change in the presence of ISO or EPI. On the other hand, in membrane fractions the level of G beta 3 significantly increased in the presence of ISO or EPI. ISO with PROP or ISO with CGP did not change the level of G beta 3 in membrane fractions. The levels of G beta 1 and G beta 2 did not change in the presence of ISO or EPI in membrane fractions. Taken together, beta-adrenoceptor agonist might induce isoform-specific translocation of G beta 3 from the cytosol to the membrane.

Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.